The Osphradium in Placopoctenmagellanicus and Pecten Maximus (Bivalvia, Pectinidae): Histology, Ultrastructure, and Implications for Spawning Synchronisation

Marine Biology (1995) 123:121-129 Springer-Verlag 1995

P. G. Beninger - A. Donval M. Le Pennec The osphradium in Placopoctenmagellanicus and Pecten maximus (Bivalvia, Pectinidae): histology, ultrastructure, and implications for spawning synchronisation

Received: 8 November 1994/Accepted: 12 December 1994

Abstract The bivalve osphradium is a band of puta- tively sensory tissue located in the gill axis, whose Introduction function is uncertain. In the present study, extending from 1987 to 1994, anatomical, histological, and elec- Most sedentary or sessile aquatic organisms present tron microscopical techniques were used to elucidate a reproductive strategy based on external fertilisation the structure and ultrastructure of the osphradium in (see Adiyodi and Adiyodi 1989, 1990). These organisms hatchery Pecten maximus L. and Placopecten magel- are also often dioecious, such that gametes are shed to lanicus (Gmelin) (collected from Passamaquoddy Bay, the external medium by male and female individuals, New Brunswick, Canada). The osphradium consists of where fertilisation success depends largely on the en- two distinct regions which run longitudinally on both counter probability of gametes. It is thus not surprising sides of each gill axis: the osphradial ridge, and the that in such organisms spawning is observed to be dorsal tuft cilia region. The osphradial ridge was large- relatively synchronous; indeed, one of the classic hatch- ly devoid of cilia other than those of the few free nerve ery techniques to induce spawning in recalcitrant female fibres. The dorsal tuft cilia region contained free nerve bivalves consists of adding male ejaculate or testicular fibres and ciliary tufts, separated by undifferentiated tissue extract to the inhalant current of the female (see epithelial cells. No paddle cilia were observed under Andrews 1979; Sastry 1979; Mackie 1984 for reviews). isosmotic fixation conditions, although under hypo- To date, however, no sensory mediator or physiological tonic conditions such cilia were quite common, sugges- pathway has been shown to be responsible for the syn- ting an artefactual nature. Most of the cells of the chronization of bivalve spawning. A recent survey of the osphradial ridge were highly secretory, the principal structure and ultrastructure of one of the enigmatic products being large pigment granules (in Pecten maxi- bivalve sensory organs has led Haszprunar (1987a) to mus) directly secreted by the Golgi bodies, and numer- propose, by careful deductive reasoning, that the os- ous small, electron-dense vesicles. These vesicles were phradium may be responsible for reception of a chemical arranged along extensive microtubule arrays in the stimulus which would induce gamete release. basal region, indicative of axonal transport. These data Enigmatic sensory organs are found in many mol- support and extend Haszprunar's hypothesis of the role luscan taxa; the principal ones in bivalves are the ab- of the osphradium in the reception of chemical spawn- dominal sensory organ and the osphradium (see Benin- ing cues and in the synchronization of gamete emission. ger and Le Pennec 1991 for reviews and references). Together with independent data on nerve pathways, The osphradium is a sensory structure assumed to have osphradial sensory modalities, and monoamine local- appeared early in the evolution of the Mollusca (Yonge isation, an anatomical pathway and neurophysiologi- 1947) and to be most highly developed in the Gas- cal mediator are postulated. tropoda. Although no function has been unequivocally assigned to this organ (Bailey and Benjamin 1968; Kraemer 1981), its position in the inhalant current of Communicated by J.P. Grassle, New Brunswick the gastropod pallial cavity originally suggested a sen- P.G. Beninger (tzc~) D6partment de Biologie, Facult6 des Sciences, Universit6 de sory role in monitoring water quality, and sediment Moncton, Moncton, N.B., E1A 3E9 Canada load in particular (Yonge 1947). Subsequent work with A. Donval M. Le Pennec gastropods indicated a chemoreceptive function (Dor- Laboratoires de Biologie Marine, Facult6 des Sciences, Universit6 sett 1986). Its relationship to the sensory structures of de Bretagne Occidentale, F-29287 Brest C6dex, France the same name in bivalves has been controversial, 122 notably due to its position in the exhalant current of washed in buffer, and post-fixed overnight in 1% osmium tetroxide at bivalves (Kraemer 1981; Haszprunar 1987a). Further- 4 ~ Initial fixations were slightly hypotonic to the external medium (by ca. 200 mOsmol); samples fixed in 1994 were isotonic. The tissue more, early anatomical studies on the bivalve os- pieces were dehydrated in an ascending ethanol series. The tissue was phradium were often misleading (see Kraemer 1981 then critical-point dried, sputter-coated with gold, and examined and Beninger and Le Pennec 1991 for discussion of this using a JEOL 100CX scanning electron microscope at 15 KV. point), generating considerable confusion. The fine morphology of the osphradium has re- cently been presented for a wide variety of molluscs TEM (Haszprunar 1985a, b; 1987a, b, 1992). Among the Bival- via, however, the family Pectinidae was absent from Portions of the gill axis containing the sensory epithelium were removed for both species, and ca. 2 mm 3 pieces were fixed in 3% Haszprunars' (1987a) review of osphradial structure and glutaraldehyde/0.1 M cacodylate (pH 7.3, adjusted to isotonic 1100 ultrastructure. Early representations were contradictory mOsmol with sucrose buffer) at 4~ post-fixed in 1% osmium and sketchy (Dakin 1909, 1910; Setna 1930). Further- tetroxide as above, dehydrated in an ascending ethanol series and more, in view of the putative role of this structure in the embedded in Spurr resin. Preliminary studies showed the tissue to be extremely sensitive to sub-optimal fixation conditions, particularly synchronisation of bivalve spawning (Haszprunar osmolarity. Semi-thin sections (1 gin) were stained using methylene 1987a), and in light of recent progress in the understand- blue (which deeply stains sensory cells). Thin sections (ca. 50 nm) ing of regulatory factors in pectinid reproduction (Paulet were contrasted using uranyl acetate and lead citrate and observed et al. 1993), it is desirable to examine the structure and using a JEOL at 50 KV. ultrastructure of the osphradium in this family. The present study reports the results of such an investigation in two pectinid species: Placopecten mageIlanicus and Pecten maximus, using histological, scanning (SEM) and transmission (TEM) electron microscopical techniques. .~:.:;- :.."

Materials and methods

Specimens .~a~.~..

Adult specimens of Placopecten mageUanicus Gmelin were collected in May 1987, September 1989, April 1994 and August 1994 from Passamaquoddy Bay (Bay of Fundy, New Brunswick, Canada). The scallops were transported in coolers to the laboratory where they ?.~g~, ~'; ~'~,i/-'~,.. were kept for several days at 6 ~ in a refrigerated recireulating seawater system. Adult Pecten maximus were transported from the Argenton hatchery (Finistare, France) to a recirculating seawater system at 13 ~ where they were kept with one algal feeding daily for 2 d prior to dissection.

Histology Fig. 1 Placopecten magellanicus, left valve removed. Note position of the osphradium (o) along the lateralmost margin of the right gill Pieces of the anterior, mid- and ventral regions of the gill axis were axis (ga). (am Adductor muscle; d9 digestive gland; 9 gill;f foot; l lips; dissected out using microsurgical instruments and processed for histo- lp labial palp.) Modified from Drew (1906) logy as previously described (Beninger 1987). The difficulty of cor- rectly locating the pectinid osphradium is a matter of historical record (Dakin 1909, 1910; Setna 1930); preliminary work on the specimens collected in 1987 indicated that it was located on the outer side of the Fig. 2 Placopecten magellanicus and Pecten maximus. Histology and gill axis only (Beninger and LePennec 1991). Accordingly, serial surface features of the osphradium. A C Placopecten magelIanicus, sections were performed at 5 tim on two specimens collected in light histology. A Location and anatomical relationships of the August 1994 within the region containing the sensory epithelium. The osphradium. (bn Branchial nerve; ga gill axis; gf gill filaments; o os- sections were alternately stained using either methylene blue or phradium.) B Detail of A, showing histology of structures associated a modification of the technique previously described (Beninger 1987), with osphradium. (av afferent branchial vessel; bn branchial nerve; in which acid fuchsin (Humason 1979) was substituted for orange-G cbn cortex of branchial nerve; ev efferent branchial vessel; np neur- and erythrosine (enhancing the contrast of muscle fibres). opil of branchial nerve; on osphradial nerve; or osphradial ridge.) C Detail of B, showing nerve tract (on, osphradial nerve) from osphradial ridge (or) to branchial nerve (cbn cortex of branchial SEM nerve). (mf subepithelial muscle fibres of the gill axis.) D-F Pecten maximus. D SEM of gill axis showing the osphradial ridge (or) and, Pieces of the gills of both species were dissected out, ensuring that ventrally, the gill filaments (gf). E detail of D, showing the osphradial the original lateral-medial orientation could be recognized by ridge (or) almost totally devoid of cilia (arrowhead). Tuft cilia (tc) are assymetrical cuts of the gill filaments, fixed overnight in 4% glutaral- found dorsally, and ordinary cilia (c) ventrally. F Detail of tuft cilia dehyde (0.1 M sodium cacodylate-NaC1 buffer, pH 7.2) at 4~ dorsal to the osphradial ridge. Note straight tips (arrowheads) 123 124 mus, contained numerous membrane-bounded pigment ResuRs granules, secreted directly by some of the Golgi bodies. These pigment granules presented a heterogeneous as- Location, morphology, and innervation pect, and were concentrated mainly in the apical region (Fig. 4A, B, C). The cytoplasm of both species was filled For clarity, the osphradium is defined in the present with granular endoplasmic reticulum and Golgi bodies, work as the sensory epithelium found on the gill axis, indicative of active secretion. Small (ca. 0.2 to 0.4 gm) whereas the underlying nerve tracts will be referred to as innervation. As will be shown below, the scallop osphradium consisted of two distinct regions: the ridge and, dorsally, the tuft cilia region. The general anatomical relationships of the scallop osphradium are shown in Fig. 1. The histological data confirmed that the osphradial ridge is situated on the mid-portion of the gill axis (Fig. 2A, B, D). It extends from the anterior region of the gill axis along the epithelium slightly ventral and parallel to the branchial nerve for approximately four-fifths the length of the gill axis, terminating in the posterior region (Fig. 1). ga Whereas in Pecten maximus a ridge of almost totally non-ciliated orange pigmented tissue constituted the ventral limit of the osphradium, this ridge was unpig- mented in Placopecten magellanicus (Fig. 2C, D,E, Fig. 3C, D, E). Serial histological sections showed that the osphradial ridge received irregular branches from the branchial nerve at intervals throughout its length (Fig. 2B, C, Fig. 3B, C); these branches can each be considered osphradial nerves. Their assymetric disposition, as well as the variably erectile state of the ridge upon fixation, are probable causes for past confusion over exact location (Setna 1930; Beninger and Le Pennec 1991).

Cytology ~L Although the two regions of the osphradium showed some similarities in cell types, marked differences in relative abundance were noted, as described below. In general, the basic cell types observed in the present study conform to the descriptions of Haszprunar (1987a) for Mytilus edulis; the following account will, therefore, recapitulate briefly and focus on cell type Fig. 3 Pecten maximus and Placopecten magellanicus gill axis. Sche- distribution and significant new information. The gen- matic representation of anatomical relationships and innervation of eral disposition of the osphradial epithelium was the osphradium. [av Afferent branchial vessel; bn branchial nerve; pseudostratified, with most cells displaying a serpentine D dorsal orientation; ev efferent branchial vessel; ga gill axis; gfgill filaments; nfc cilium of free nerve fibre on osphradial ridge (or); on configuration much like that of the human olfactory one of the osphradial nerves; tc tuft cilia; V ventral orientation] epithelium (Kessel and Kardon 1979), which usually resulted in truncated sections for TEM observation.

Fig. 4 Pecten maximus osphradial ridge. A Secretory cells (sc) com- Osphradial ridge prising pseudostratified epithelium. (my microvilli; n nucleus; Pg pigment granules.) B Detail of A, showing endoplasmic reticulum Along with occasional mucocytes, the osphradial ridge (er) and numerous electron-dense secretion granules (sg). C mid- comprised a pseudostratified layer of very active se- region of a secretory cell, showing extensive granular endoplasmic reticulum (rer), several Golgi bodies (G), as well as pigment granules cretory cells, traversed at rare intervals by free nerve (pg) and numerous electron-dense secretion granules (sg). D and fibres (Fig. 4A, B, Fig. 5A, B). The secretory cells pos- E Basal region of a secretory cell, showing arrangement of secretion sessed short (ca. 5 gm) microvilli, and, in Pecten maxi- granules along microtubules (mr) 125 126 127 electron-dense secretion granules were extremely abun- allow it to blend in with the general gill axis epithelium dant, and in the basal region they aligned with the when histologically processed and stained using extensive, parallel microtubule array which extended topological protocols; the irregular innervation com- toward the basal innervation from the osphradial pounds the problem. In contrast to the original descrip- nerves (Fig. 4B, C, D, E). The nuclei were irregular, and tion of Dakin (1910), and in agreement with that of contained peripheral and dispersed heterochromatin Kraemer (1981), the cells of the osphradial epithelium (Fig. 4A, B, Fig. 5A). are not triangular; in both scallop species examined the The rare free nerve fibres found in the osphradial cells were highly variable and serpentine in shape. ridge are characterized by the presence of few cilia, Unreported to date is the spatial separation of the pseudopodia-like extensions rather than microvilli in osphradium into two discrete components: the sparsely the apical region, and parallel nerve processes extend- ciliated osphradial ridge and the dorsally adjacent tuft ing the length of the cell toward the basal innervation cilia region. This anatomical separation is accom- (Fig. 5A, B). Secretory structures such as Golgi bodies, panied by a cytological specialization of each region. vesicles, and rough endoplasmic reticulum (RER) were The ultrastructural characteristics of the cell types of rare. Nuclei were not observed and were assumed to be the tuft cilia region correspond to those reported by subepithelial. Haszprunar (1987a) for Mytilus edulis, although no paddle cilia were observed under the isotonic fixation conditions of the present study. It is probable that Tuft cilia region paddle cilia are artefacts of hypotonic fixation (Short and Tamm 1989, 1991; Deiner and Tamm 1991; Deiner The tuft cilia region contained two distinct types of et al. 1993; Beninger et al. in press). neurosensory elements: cells terminating in a dense tuft The variety of functions proposed for the os= of cilia (Fig. 2E, Fig. 5D) and free nerve fibres as de- phradium were summarized by Haszprunar (1987a). scribed above (Fig. 5E). The free nerve fibres were much Considering the near-total lack of experimental data, more numerous in this region compared to the os- the most convincing deductive arguments were pre- phradial ridge. sented in the above-mentioned paper. Based on his The tufts of cilia were separated by nonciliated, un- extensive survey of osphradial structure in the Mol- differentiated epithelial cells (Fig. 2E, Fig. 5E), also lusca, Haszprunar (1987a) argued for a chemosensory termed supporting cells (Haszprunar 1987a). The tuft role primitively associated with external fertilisation, cilia cells conformed to the description of "paddle cilia" which then evolved into food detection in taxa which cells (Haszprunar 1987a), although paddle cilia were acquired internal fertilisation. As bivalves all rely on only observed in tissues which had been hypotonically some form of gamete broadcast for fertilisation, his fixed in initial preparations. Due to the electron density hypothesis of a chemosensory role in detecting spawn- of the cytoplasm, it was not possible to trace the course ing cues from conspecifics would apply to this class of of the ciliary roots. However, numerous nerve pro- molluscs. Although a central argument concerned the cesses connected the base of the tuft cilia region to the presence of paddle cilia, chemosensory function in base of the osphradial ridge (Fig. 5C). invertebrates may be accomplished by cilia with no morphological distinguishing features (Laverack 1968, 1988). Chemoreception has in fact been demon- Discussion strated in the osphradium of two freshwater bivalves, Unio pectorum and Anodonta cygnea (Sokolov and Setna (1930) emphasized the cryptic nature of the pec- Zaitseva 1982). tinid osphradium, which, due to its variable degree of Of particular interest is the demonstration of the retraction upon fixation, may not appear in SEM ob- highly secretory nature of most of the cells comprising servation. Similarly, the retraction and compactness the epithelium of the osphradial ridge. Although pigment granules are common in the cells of the osphradium (Haszprunar 1985a, b, 1987a, b), these large inclusions were seen in the present study to be secreted Fig. 5 Pecten maximus. Transmission electron microscopy of os- whole by Golgi bodies in Pecten maximus. In contrast, phradial ridge and adjacent tuft cilia region. A One of the rare free the dense concentration of small secretion vesicles and nerve fibres in the osphradial ridge, containing nerve processes (fnp) and adjacent secretory cell (sc). The free nerve fibre cell (nfc) posesses RER attest to the secretion of another discrete product; one cilium. (my microvilli; n nucleus.) B Detail of apical region of the size and density of these granules are typical of a free nerve fibre cell, showing nerve processes (np) extending toward neurosecretory granules found throughout the animai cell base. C Transverse section of basal portion of free nerve fibre cell kingdom (Coggeshall 1967). Similar profiles have in the tuft cilia region, showing extensive nerve processes (rip). been reported in gastropods (Coggeshall 1967; Boer et D Transition between epithelia of osphradial ridge (or) and dorsal tuft cilia region (tcr). (my microvilli.) E Epithelium of tuft cilia region, al. 1968; Bonga 1970; Crisp 1971), as well in the acces- showing tuft cilia arising from ciliated cell (cc) and adjacent free sory lobe of the parietovisceral ganglion of Pecten nerve fibre cell (fnp) and undifferentiated (supporting) cell (uc) maximus (A. Donval unpublished), known to be rich in 128 monoamines (Paulet et al. 1993). In molluscs, the pro- contributing to or otherwise influencing the axonal duction of neurosecretions involves both the granular transport of serotonin-like monoamines to the gonad endoplasmic reticulum and Golgi bodies (Dorsett via the gonad nerve (Fig. 6). These monoamines induce 1986), both of which were very abundant in these the release of gametes, possibly in response to chemical secretory cells. Furthermore, both the density of micro- cues in the ejaculate of neighboring conspecifics. tubules in the basal region of these cells and the The results of the present study, together with the alignment of the small secretion vesicles along the micro- foregoing considerations, thus suggest a role of the tubules are hallmarks of axonal transport of neurotrans- scallop osphradium in the synchronization of spawning mitters/neurohormones in molluscs (Dorsett 1986). in bivalves, as originally proposed by Haszprunar Measurements of "fast" axonal transport (i.e., along (1987a). However, Haszprunar suggested that the os- microtubules) give values of 1 to 2 gm s - 1, or ca. 1.5 cm phradium responded to immediate spawning cues con- in 3 h, which is far too slow to provide immediate tained in the spawning products of conspecifics, reaction to stimuli (Schroer and Kelly 1985; Vale et al. especially males, corresponding to the well-known phe- 1985 a, b, c). However, such transport could be involved nomenon of facilitation of spawning in females by the in the accumulation of neurosecretions for reaction to addition of male ejaculate to their inhalent current a forthcoming stimulus. The anatomical pathway of the (Andrews 1979; Sastry 1979; Mackie 1984). The present osphadial innervation is to the osphradial nerves, join- study suggests that the osphradium may be involved in ing the branchial nerve, which sends a branch to the both preparatory (transport and accumulation of neu- accessory lobe of the parietovisceral ganglion. Studies rosecretions in the accessory lobe) and immediate (re- of the parietovisceral ganglion of Pecten maximus using lease of stored monoamines to the gonad) responses. monoclonal antibodies have demonstrated the localisa- The preceding arguments suggest that spawning tion of serotonin-like monoamines in the accessory synchronisation is achieved by successive refinement lobe only (Paulet et al. 1983). Serotonin is a potent of maturation in response to environmental cues. spawning inducer in bivalves, including pectinids (Mat- Temperature and food availability are important con- sutani and Nomura 1982; Gibbons et al. 1983; Gibbons ditioners for approximate synchrony, but precise syn- and Castagna 1984). The only other nerve which con- chrony may rely upon two chemical cues from neigh- nects to this lobe is the gonad nerve (Fig. 6). boring conspecifics: an initial "ready" cue in the days or We thus postulate that chemical spawning cues emit- hours preceding spawning, and a final "go" cue upon ted by conspecifics in the penultimate stages of matura- commencement of spawning. tion are detected by the osphradial sensory cells, induc- It is evident that behavioural and electrophysiologi- ing a neurosecretory activity in the cells of the os- cal experimentation should be undertaken to further phradial ridge; neurosecretions are transported to investigate this hypothesis. In particular, stimulation of the accessory lobes of the parietovisceral ganglion, the osphradium in mature bivalves should be at- tempted in order to determine whether this can influ- ence spawning. Two major obstacles to such work are the cryptic anatomical character of the bivalve osphradium, which is difficult to localize in vivo for most species, and the inaccessibility of this structure in undis- turbed bivalves. Use of a combined endoscope (Ward et al. 1991) and submersible electrode might provide an avenue of investigation.

Acknowledgements The authors extend thanks to MM. A. LeMer- cier and L Blanchard for their professional photographic assistance. The scientific drawings of Figs. 2 and 6 were kindly provided by Mad. S.D. St-Jean, for which we are most grateful. This research was supported by operating grants from the Facult~ d'Etudes Sup~rieures et de la Recherche, Universit~ de Moncton, and the Natural Sciences and Engineering Research Council of Canada.

Fig. 6 Pecten rnaximus. Schematic drawing (after Dakin 1910) of the parietovisceral ganglion, showing pathway from osphradium via the branchial (bn) and osphradio-branchial nerves to the accessory lobe References (a/). Although no osphradio-branchial nerve (obn) was observed in the gill axis, it is reported in the region of the parietovisceral Adiyodi KG, Adiyodi RG (1989) (eds) Reproductive biology of ganglion by Dakin (1910). The gonad nerve (gn) leaves the accessory invertebrates, Vol. IV, Part A. Fertilization, development and lobe to innervate the gonad (go). Stippled regions, the accessory parental care. John Wiley and Sons, Chichester lobes, show areas of the parietovisceral ganglion which gave positive Adiyodi KG, Adiyodi RG (1990) (eds) Reproductive biology of reaction to monoclonal antibodies for serotonin (Paulet et al. 1993). invertebrates, Vol. IV, Part B. Fertilization, development and (cl central lobe; cvc cerebro-visceral connective; lI lateral lobe) parental care. John Wiley and Sons, Chichester 129

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